Source data are provided with this paper. Abstract Immune checkpoint blockade (ICB) therapy does not benefit the majority of treated patients, and those who respond to the therapy can become resistant to it. 1 (PD1) antibodies for the monitoring of antitumour responses to ICB Rabbit Polyclonal to AML1 therapy. The sensors consist of a library of mass-barcoded protease substrates that, when cleaved by tumour and immune proteases, are released into urine, where they can be detected by mass spectrometry. By using syngeneic mouse models of colorectal cancer, we show that random-forest classification trained on mass-spectrometry signatures from a library of PD1-conjugated mass-barcoded activity sensors for BMX-IN-1 differentially expressed tumour and immune proteases can be used to detect early antitumour responses and to discriminate resistance to ICB therapy driven by loss-of-function mutations in either the B2m or Jak1 genes. Our data supports the use of activity-based biomarkers for early on-treatment response assessment and classification of refractory tumours based on resistance mechanisms. One-sentence editorial summary: A library of systemically administered protease-cleavable sensors conjugated to anti-programmed cell death protein 1 antibodies allows for the early urinary detection and monitoring of antitumour responses to immune checkpoint blockade therapy in mice. Immune checkpoint blockade (ICB) therapy has transformed the treatment of cancer for patients across a broad range of malignancies1,2. ICB involves the administration of antibodies that block inhibitory checkpoint molecules, such as the cytotoxic T lymphocyte-associated protein 4 (CTLA4) or the programmed cell death protein 1 (PD1), to reinvigorate an antitumour T cell response. Despite the potential for ICB to produce durable clinical outcomes, a large fraction of patients do not derive clinical benefit1,3. Objective response rates remain below ~25% in many cancer types, largely due to immunosuppressive factors in the tumour microenvironment (e.g., regulatory T cells or myeloid-derived suppressor cells) and primary tumour-intrinsic mutations1. In addition, responsive tumours can acquire resistance during therapy such as in metastatic melanoma where up to one-third of patients with initial responses to ICB therapy eventually relapse3. Both primary and acquired resistance are driven by mechanisms that enable tumour cells to evade antitumour immune responses, including defects in antigen presentation or in the interferon gamma (IFN) response pathway3,4. Therefore, developing noninvasive biomarkers of immune response and resistance to ICB has emerged as a clinical priority5. Patient responses to ICB therapy are currently assessed using a combination of radiographic, tumour, and serum biomarkers5. Radiographic evaluation by Response Evaluation Criteria in Solid Tumours BMX-IN-1 (RECIST) is the standard assessment method and occurs after the first cycle of ICB therapy, which consists of BMX-IN-1 3C4 doses administered within an 8C12-week window6C8. The observation of atypical patterns of response to ICB has motivated continual refinement to the timing and frequency of radiographic assessment such as the development of immune-related response criteria (e.g., irRC, irRECIST) to account for phenomenon like pseudoprogression5,9. Tumour biomarkers such as programmed death-ligand 1 (PD-L1) expression have been shown to enrich for populations with clinical benefit, but have limitations as predictive biomarkers as at least ~40C50% patient tumours with PD-L1 positivity do not experience objective responses5,10. Other tumour biomarker strategies, such as assessing on-treatment changes in tumour mutational burden by whole exome sequencing11, are BMX-IN-1 promising and have been found to correlate with PD1 response. However, these approaches require serial biopsies, which in practice are not typically collected over the course of therapy with attendant patient risks. Therefore, considerable interest is focused on identifying noninvasive biomarkers to allow longitudinal and quantitative assessment. These include quantifying changes in T cell clonality or circulating tumour DNA levels, which have been shown to be detectable within 3C4 weeks of treatment and correlate with objective response and overall survival12C14. These studies highlight the considerable interest and need for noninvasive and longitudinal assessment strategies to track response and resistance to ICB therapy early on-treatment. Proteases BMX-IN-1 play fundamental roles in cancer biology, immunity, and antitumour responses and therefore may provide a mechanism to evaluate ICB therapy. Tumour-dysregulated proteases (e.g., matrix metalloproteases, cathepsins) are involved in proteolytic cascades that change the tumour microenvironment during angiogenesis, growth, and metastasis15,16. In addition, T cell-mediated.